Dried spore germinative compound mixtures

ABSTRACT

In one aspect, the present invention is directed to a dried intimate mixture comprising a bacteria spore and a germinative compound, and methods for preparing the intimate mixture. In another aspect, this invention is directed to a composition comprising such an intimate mixture. The invention also relates to methods for increasing the germination, growth, metabolism, and/or enzyme activity of a bacteria spore comprising preparing an intimate mixture of a bacteria spore and a germinative compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 61/849,973 filed Feb. 6, 2013, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

In one aspect, the present invention is directed to a dried intimatemixture comprising a bacterial spore and a germinative compound, andmethods of preparing the mixture. In another aspect, this invention isdirected to a composition comprising such a mixture. The invention alsorelates generally to methods for increasing the germination, growth,metabolism and/or enzyme activity of bacteria spores, comprisingpreparing a dried intimate mixture comprising a bacterial spore and agerminative compound.

BACKGROUND OF THE INVENTION

The use of spore forming bacteria including certain Bacillus strains asprobiotics for both humans and animals has become prevalent in recentyears. As is noted in Knap et al. (WO 2010/070005) species such asBacillus subtilis and Bacillus licheniformis are used as supplements inanimal feed in order to promote growth by increasing the digestion andavailability of nutrients from animal feed. Bacillus coagulans is theactive ingredient in commercial probiotic products for humanconsumption, helping to aid in the digestion of proteins, lactose andfructose.

As is noted in Maathuis et al. (2010, Beneficial Microbes, 1(1): 31-36),such bacteria must be present in the small intestine in their germinatedor vegetative form in order to function as probiotics. While suchmicrobes are resistant to both stomach acid and bile salts in theirspore form, they are susceptible to such environments in theirvegetative states. Thus, if employed in their vegetative state, Bacillusstrains must be contained within a pharmaceutically-acceptableacid-resistant or “enteric” carrier. See paragraph 7 of Farmer (USPatent Application 2003/0124104).

Unfortunately, it is difficult to formulate Bacillus species in theirvegetative form such that they will possess an adequate shelf life. Asis noted in GanedenBC product literature, traditional vegetativeprobiotics do not survive high heat and pressure in the manufacturingprocess, die quickly on the shelf, and are sensitive to stomach acidsand bile enzymes in the gut. In contrast, formulations of such speciesin their spore form are much more suitable for commercial and practicaluse. Thus, as is noted by Cartman et al. (2008, Applied andEnvironmental Microbiology, August, p. 5254-5258) “[b]acterial sporesare particularly well suited for use as live microbial products as theyare metabolically dormant and highly resilient to environmental stress.These intrinsic properties are highly desirable from a commercialperspective and mean that spore-based products have a long shelf lifeand retain their viability during distribution and storage.”

The use of certain compounds, particularly certain L-amino acids, tostimulate the germination of Bacillus spores has been reported in theliterature. Thus, for example, Foerster et al. (1966, Journal ofBacteriology 91(3): 1168-1177) discloses that the addition of L-alanineto spore suspensions in aqueous solutions will cause the germination ofa number of Bacillus species. In addition, Maathius et al. cited above,suggests that the Bacillus coagulans spores in GanedenBC could betriggered into germination at the beginning of the small intestine byingesting them together with a diet containing L-alanine, or byincluding L-alanine with such spores in a powder formulation. However,the approaches suggested by Maathius present several major challenges toestablishing a probiotically effective bacterial culture:

1) Although the Bacillus coagulans spores employed in GanedenBC arethemselves largely resistant to the low pH in the stomach, exposure tosuch acids could lead to a lag in germination when such spores enterinto a more neutral pH. For example, Blocher et al. (1985, Applied andEnvironmental Microbiology 50(2): 274-279) demonstrated that B. cereusspores were inhibited from germinating at pH 4.5 even in the presence ofthe germinative compounds L-alanine or L-cysteine. Spores sequentiallyexposed to pH 4.5 buffer followed by pH 7.0 buffer were able togerminate upon exposure to such L-amino acids, but exhibited a lag incommitment to germinate. Any substantial delay in germination is highlyundesirable, given the relatively short period of time that the sporesmay be present in the small intestines before being excreted. This isparticularly true in smaller animals such as chicks, which have feedtransit times of about 1.5 hours when 1 day old and transit time of lessthan 2 hours when 7 days old (see B. C. Watson et al. (2006, PoultryScience 85: 493-497), and shrimp, which have a transit time of less than90 minutes (see Beseres et al., 2005, Journal of Shellfish Research24(1):301-308). Thus there remains a need for accelerating andincreasing germination of bacterial spores under conditions of exposureto low pH, such as those found in the stomach.

2) Diets high in L-alanine may also be high in D-alanine. As noted byAtluri et al. (2006, Journal of Bacteriology 188(1): 28-36), and Blocheret al. (cited above), D-alanine is a powerful inhibitor of Bacillusgermination. In addition, there can be large amounts of othergermination inhibitors (e.g., other D-amino acids, inorganic and organicacids, fatty acids, and bile salts) present in the small intestine whichcould compete with L-alanine if mixed in a powder foam with Bacillusspores. Thus, a need exists to develop a method of improving sporegermination in the presence of germination inhibitors that may competewith germinative compounds.

Accordingly, it is an object of this invention to provide a bacterialspore formulation which is capable of providing such benefits.

SUMMARY OF THE INVENTION

It has been surprisingly found that bacterial spore germination, growth,metabolism and enzyme activity are increased through formation of anintimate mixture of the bacterial spore and a germinative compound.Unexpectedly, the intimate mixture also increases germination and growthof the bacteria spores in the presence of germination inhibitors.

In one aspect, the present invention is directed to a dried intimatemixture comprising a bacterial spore and a germinative compound, whereinthe bacteria spore and the germinative compound are maintained inproximate position until they reach an environment conducive togermination.

In another aspect, the present invention is directed to a compositioncomprising a dried intimate mixture comprising a bacterial spore and agerminative compound, wherein the bacteria spore and the germinativecompound are maintained in proximate position until they reach anenvironment conducive to germination.

In a further aspect, the present invention relates to a method forpreparing a dried intimate mixture comprising a bacteria spore and agerminative compound, the method comprising:

a) preparing a solution comprising a bacteria spore and a germinativecompound; andb) drying the solution to obtain a dried intimate mixture comprising abacteria spore and a germinative compound,wherein the bacteria spore and the germinative compound are maintainedin proximate position until they reach an environment conducive togermination

In a still further aspect, the present invention relates to a method forincreasing the germination, growth, metabolism and/or enzyme activity ofbacteria spores, comprising:

a) preparing a solution comprising a bacteria spore and a germinativecompound; andb) drying the solution to obtain a dried intimate mixture comprising abacteria spore and a germinative compound, wherein the bacteria sporeand the germinative compound are maintained in proximate position untilthey reach an environment conducive to germination of the bacterialspores; andc) exposing the intimate mixture to an environment conducive togermination of the bacteria spores, wherein the germination, growth,metabolism and/or enzyme activity of the bacteria spores in the intimatemixture is increased relative to a corresponding bacteria sporeformulation that lacks a germinative compound.

DETAILED DESCRIPTION OF THE INVENTION

The mixtures of this invention are comprised of a bacterial spore and agerminative compound. The bacterial species employed may be any specieswhich forms spores, and is typically one which exhibits desirableprobiotic activity in humans or animals. However, bacterial specieswhich have other industrial applications including those useful inagriculture, environmental remediation, composting or methaneproduction, cleaning supplies and the like may also be employed. Suchspecies include spore forming members of the phylum Firmicutes and sporeforming members of the phylum Actinobacteria as listed in Bergey'sManual of Systematic Bacteriology, Second Edition (2009), herebyincorporated by reference in its entirety. Members of the phylumFirmicutes include aerobic spore-forming species (generally previouslydefined as Bacillus species) and anaerobic spore-forming species(generally previously defined as Clostridium species). Illustrativespecies of the phylum Firmicutes include B. alcalophilus, B. alvei, B.amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B.atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B.centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B.firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, Blarvae, B. laterosporus, B. lentimorbus, B. lentus, B. licheniformis, B.megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B.pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B.pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans,B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B.thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C.ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuriapenetrans, Pasteuria thornei, and Pasteuria nishizawa, as well asgenetically modified variants of such species. Preferred species includeB. amyloliquefaciens, B. brevis, B. cereus, B. circulans, B. clausii, B.coagulans, B. firmus, B. laterosporus, B. lentus, B. licheniformis B.megaterium, B. polymyxa, B. pumilus, B. simplex, B. sphaericus, B.stearothermophilus, B. subtilis, B. thuringiensis and C. butyricum.Members of the phylum Actinobacteria include Streptomyces species, aswell as genetically modified variants of such species. Preferred speciesinclude Streptomyces viridochromogenes, Streptomyces griseoviridis,Streptomyces lydicus, Streptomyces plicatus, Streptomyces sindeneusis,Streptomyces rochei, Streptomyces alni, Streptomyces viridis,Streptomyces thermovulgaris, Streptomyces griseus, Streptomycesacidiscabies, Steptomyces aureofaciens, Streptomyces galbus,Streptomyces microflavus, and Streptomyces aureofaciens.

The germinative compound may comprise any compound which is effective tocause germination of the particular spore-forming bacteria species withwhich it is intimately mixed and which is amenable to processes usefulfor forming such an intimate mixture such as spray-drying,freeze-drying, air drying or drum drying. Typically, such germinativecompounds are L-amino acids including L-alanine, L-valine, L-proline,L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine,L-phenylalanine and analogues thereof. Such analogues can be created byone of ordinary skill in the art by making substitutions on or withingroups of a base chemistry. Thus, for example, analogues of L-alanineinclude: L-Leu-L-Ala, L-Ala-L-Leu, L-Pro-LAla, L-Ala-L-Pro,a-L-Glu-L-Ala, L-Ala-L-Glu, L-His-L-Ala, L-Ala-L-His, L-Ala-L-Ala,Gly-L-Ala, L-Ala-Gly, N42-methylsulphonyl)ethyloxycarbonyl-L-alaninedicyclohexylammonium salt (N-MSOC-L-Ala), N-t-butoxycarbonyl-L-alanine(N-t-BOC-L-Ala), N-acetyl-L-alanine (N-Ac-L-Ala),N-2,4-dinitrophenyl-L-alanine (N-DNP-L-Ala), Ncarbobenzoxy-L-alanine(N-CBZ-L-Ala), 5-dimethylamino-1-naphthalenesulphonyl-L-alaninecyclohexylamine salt (N-dansyl-L-Ala), N-benzoyl-L-alanine (N-Bz-L-Ala),L-alanine methyl ester hydrochloride, L-alanine ethyl esterhydrochloride, L-alanine t-butyl ester hydrochloride, L-alanine benzylester hydrochloride, L-alaninamide, L-alanine p-nitroanilidehydrochloride and L-alaminol. In one embodiment, the germinativecompound is an amino acid selected from the group consisting ofL-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine,L-glutamine, L-asparagine, and L-phenylalanine. Preferably, suchgerminative compound is L-alanine.

Such amino acids can be employed as individual compounds, or in the formof polypeptides. In one embodiment the polypeptides are proteinhydrolysates, for example casein hydrolysate. Useful polypeptides willtypically comprise at least fifty percent amino acids which willfunction as germinants, such as L-alanine, L-valine, L-proline,L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine, andL-phenylalanine, such percentages being based upon the number of aminoacids in the polypeptide.

In a preferred embodiment, the germinative compound is selected from thegroup consisting of L-amino acids, proteins, sugars, and salts.Particularly preferred bacterial spore/germinative compound combinationsinclude

L-alanine+Bacillus subtilis, L-alanine+Bacillus licheniformis,L-alanine+Bacillus pumilus, L-alanine+Bacillus amyloliquefaciens,L-alanine+Bacillus coagulans, L-alanine+Bacillus cereus,L-alanine+Bacillus clausii, L-alanine+Clostridium butyricumL-valine+Bacillus subtilis, L-valine+Bacillus licheniformis,L-valine+Bacillus pumilus, L-valine+Bacillus amyloliquefaciens,L-valine+Bacillus coagulans, L-valine+Bacillus cereus, L-valine+Bacillusclausii, L-valine+Clostridium butyricumL-alanine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillusamyloliquefaciens, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus coagulans, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus cereus, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus clausii, L-alanine+glucose+fructose+potassium ions(GFK)+Clostridium butyricumL-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacilluslicheniformis, L-asparagine+glucose+fructose+potassium ions(GFK)+Bacillus pumilus, L-asparagine+glucose+fructose+potassium ions(GFK)+Bacillus amyloliquefaciens,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus cereus,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus clausii,L-asparagine+glucose+fructose+potassium ions (GFK)+Clostridium butyricumL-alanine+inosine+Bacillus subtilis, L-alanine+inosine+Bacilluslicheniformis, L-alanine+inosine+Bacillus pumilus,L-alanine+inosine+Bacillus amyloliquefaciens, L-alanine+inosine+Bacilluscoagulans, L-alanine+inosine+Bacillus cereus, L-alanine+inosine+Bacillusclausii, L-alanine+inosine+Clostridium butyricum,L-proline+glucose+Bacillus megaterium, L-proline+Bacillus megaterium,and L-lactate+Clostridium butyricum.

In certain embodiments, preferred mixtures of the present inventioninclude those wherein the spore is selected from the group consisting ofB. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium, andB. pumilus; and the germinative compound is selected from the groupconsisting of L-alanine, L-valine, and L-asparagine.

The germinative compound is present in an amount sufficient to cause thebacterial spore employed to germinate. Although this can be readilydetermined for any particular bacterial spore/germinative compoundmixture by routine experimentation, such germinative compounds aretypically formulated, prior to drying, at concentrations of from 0.0001mg/mL to 170 mg/mL. In some embodiments, the germinative compounds areformulated, prior to drying, at concentrations of 0.0003 mg/mL to 170mg/mL, 0.0003 mg/mL to 30 mg/mL, 0.001 mg/mL to 100 mg/mL, or 0.001mg/mL to 10 mg/mL. In a preferred embodiment, the germinative compoundis formulated, prior to drying, at concentrations of from 0.001 mg/mL to1 mg/mL.

As is employed herein, the term “intimate mixture” refers to a mixturein which the spores and germinative compounds are maintained inproximate position until they reach an environment conducive togermination.

Such an intimate mixture can be achieved employing processes such asspray-drying, freeze-drying, air drying or drum drying. In a preferredembodiment, the intimate mixture is produced by spray-drying orfreeze-drying. When forming such intimate mixtures, it is important thatthe spore and germinative compound are not mixed together underconditions which would allow the germinative compound to cause the sporeto germinate, as this could cause premature germination with an adverseeffect upon the storage life of the mixture. This can be avoided byemploying separate streams in a spray-dryer, either by using two nozzlesor a single nozzle which permits the simultaneous spraying of twoseparate streams; or by freeze-drying under conditions (for exampletemperatures) which are not conducive to germination. Prematuregermination may also be avoided by introducing the spore mass to asolution containing the germinative compound immediately prior todrying.

In a preferred embodiment, the germinative compound is adsorbed to orabsorbed by the bacteria spore in the intimate mixture. In a furtherembodiment, the bacteria spore and germinative compound are finelydispersed throughout the intimate mixture. In a still furtherembodiment, the bacteria spore and germinative compound aremicroscopically dispersed throughout the intimate mixture, such thatindividual particles consisting essentially of bacteria spores andindividual particles consisting essentially of germinative compounds arenot visible to the naked eye.

In one embodiment the intimate mixture is prepared by combining thebacteria spore and germinative compound in a solution prior to drying.Preferably, the bacteria spore and germinative compound are combined ina solution immediately prior to drying.

Although not wishing to be held to any theory, it is believed that theformation of an intimate mixture places the germinative compound in aproximate position where it can more preferably bind to the germinationinitiator sites of the spore when the mixture reaches an appropriateenvironment for germination. Such proximate position permits thegerminative compound to outcompete germination interfering compounds(such as D-amino acids) which may be present, with the result that ahigher percentage of spores will be germinated. Due to the logarithmicgrowth of bacteria once they enter the vegetative stage; such anincreased percentage can quickly result in a several log increase inculture formation.

The present invention also relates to compositions comprising anintimate mixture comprising (a) a bacterial spore and (b) a germinativecompound, wherein the spore and germinative compound are maintained inan inactive form such that the germinative compound will not inducegermination of the spore until such composition is subjected to anactivation environment.

As is employed herein, the term “activation environment” refers to anenvironment which permits the germinative compound and the spore tointeract, such that the spore is induced to enter into a vegetativestate. Such an activation environment may involve a combination offactors such as temperature, moisture, pH or salinity.

The formation of an intimate mixture of a germinative compound and abacteria spore may enhance the utility of the bacteria spore byincreasing one or more traits of interest of the bacteria spore,including but not limited to germination, growth, metabolism, enzymeactivity and tolerance to environmental stresses such as low pH, highsalt concentration, and exposure to toxic metals. In some embodiments,the intimate mixture increases the trait of interest by at least 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500% relative to acorresponding bacteria spore formulation that lacks a germinativecompound.

In order to further enhance germination, it is preferred that the sporebe shocked prior to the formation of the intimate mixture. Spores may beshocked in a variety of standard methods, e.g., osmotic shock, heatshock, pressure shock, deprivation of nutrients, and/or exposure tocertain acids. Heat shock involves heating the spores for a sufficientperiod of time at a temperature sufficient to induce the production ofheat shock proteins. Atluri et al., cited above, describes one such heatshock treatment for Bacillus subtilis.

The compositions of this invention comprise an intimate mixturecomprising a bacteria spore and a germinative compound. Suchcompositions may further comprise additional components, includingco-germinants, nutrients, and formulation aids (for example surfactantsand/or enteric coatings) depending upon their intended use.

Co-germinants which may be employed include purine nucleosides such asinosine or adenosine, salts, sugars (such as glucose and fructose), andthe like; all of which are well known to those of skill in the art.

Nutrients, including dextrose, starches, and micronutrients which willaid in the multiplication of bacterial colonies once the spores havegerminated may also be included.

When the composition is intended for use as a probiotic, the use of anenteric coating is preferably employed in order to avoid the lag inspore germination associated with the exposure of spores to low pHenvironments. Such enteric coating is designed to resist solution in thestomach and to dissolve in the neutral or alkaline intestinal fluid.Such coating may be pH-sensitive, e.g., not dissolving in an acidicenvironment as is encountered in the stomach but dissolving in a neutralenvironment as is encountered in the small intestine. Alternatively, theenteric coating may dissolve when exposed to specific metabolic eventsuch as an encounter with a digestive enzyme that is found in the smallintestine. For example, the coating is digested by a pancreatic enzymesuch as trypsin, chymotrypsin, or a pancreatic lipase. Digestion ordissolution of the coating allows the Bacillus spore/germinativecompound to enter into an environment conducive to the germination ofthe spores.

Enteric coating materials which may be employed are known in the art andinclude alginates, malic acid-propane 1,2-diol; cellulose derivatives,e.g., cellulose acetate phthalate or hydroxypropyl methylcellulosephthalate (HPMCP); cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropylmethylcellulose phthalate and anionic polymersof methacrylic acid and methyl methacrylate; and a water emulsion ofethylacrylate methylacrylic acid copolymer, or hydroxypropyl methylcellulose acetate succinate (HPMAS).

When employed in agricultural or industrial uses, such compositions mayfurther comprise standard formulation aids such as surfactants,emulsifiers, other active ingredients, etc. so long as such othercomponents do not interfere with germination or adversely affect theviability of the germinated spores. For example, phenolic compoundswhich are not otherwise particularly sporocidal are known to inhibitgermination at concentrations as low as 0.2% (phenol), 0.08% (cresol),and 0.02% (chlorocresol) (wt./vol). Other compounds which may inhibitgermination are also well known in the art. See, for example, A. D.Russell, Bacterial Spores and Chemical Sporicidal Agents, CLINICALMICROBIOLOGY REVIEWS, April 1990, p. 99-119.

The invention also provides a method for preparing a dried intimatemixture comprising a bacteria spore and a germinative compound, themethod comprising:

a) preparing a solution comprising a bacteria spore and a germinativecompound; andb) drying the solution to obtain a dried intimate mixture comprising abacteria spore and a germinative compound, wherein the bacteria sporeand the germinative compound are maintained in proximate position untilthey reach an environment conducive to germination.

In a preferred embodiment the drying in the aforementioned method isspray-drying, freeze-drying, air drying or drum drying. In anotherpreferred embodiment, the spore in the aforementioned method is selectedfrom the group consisting of B. alcalophilus, B. alvei, B.amyloliquefaciens, B. aneurinolyticus, B. anthracia, B. aquaemaris, B.atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B.centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B.firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, Blarvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B.megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B.pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B.pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans,B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B.thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C.ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuriapenetrans, Pasteuria thornei, Pasteuria nishizawae, and Streptomycesspp.

In a further preferred embodiment, the spore in the aforementionedmethod is selected from the group consisting of B. subtilis, B.amyloliquefaciens, B. licheniformis, B. megaterium, and B. pumilus; andthe germinative compound in the aforementioned method is selected fromthe group consisting of L-alanine, L-valine, and L-asparagine.

The germinative compound in the aforementioned method may be formulated,prior to drying, at concentrations from 0.0001 mg/ml to 170 mg/ml. Insome embodiments of the aforementioned method, the germinative compoundsare formulated, prior to drying, at concentrations of 0.0003 mg/mL to170 mg/mL, 0.0003 mg/mL to 30 mg/mL, 0.001 mg/mL to 100 mg/mL, or 0.001mg/mL to 10 mg/mL. In a preferred embodiment of the aforementionedmethod, the germinative compound is formulated, prior to drying, atconcentrations of from 0.001 mg/mL to 1 mg/mL. In certain embodiments,the germinative compound in the aforementioned method is a polypeptide.In a preferred embodiment of the aforementioned method, the spore hasbeen shocked.

The invention also provides a dried intimate mixture produced by theaforementioned methods.

In another aspect, the invention provides a method for increasing thegermination, growth, metabolism and/or enzyme activity of bacteriaspores, comprising:

a) preparing a solution comprising a bacteria spore and a germinativecompound; andb) drying the solution to obtain a dried intimate mixture comprising abacteria spore and a germinative compound, wherein the bacteria sporeand the germinative compound are maintained in proximate position untilthey reach an environment conducive to germination of the bacterialspores; andc) exposing the intimate mixture to an environment conducive togermination of the bacteria spores, wherein the germination, growth,metabolism and/or enzyme activity of the bacteria spores in the intimatemixture is increased relative to a corresponding bacteria sporeformulation that lacks a germinative compound.

In some embodiments of the aforementioned method, the intimate mixtureincreases the trait of interest by at least 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400 or 500% relative to a correspondingbacteria spore formulation that lacks a germinative compound.

In a preferred embodiment of the aforementioned method, the percentgermination, growth, metabolism and/or enzyme activity of bacteriaspores in the intimate mixture is increased by at least 10% relative tothe corresponding bacteria spore formulation that lacks a germinativecompound. In a further preferred embodiment of the aforementionedmethod, the drying is spray-drying, freeze-drying, air drying or drumdrying.

In certain embodiments, the spore in the aforementioned method isselected from the group consisting of B. alcalophilus, B. alvei, B.amyloliquefaciens, B. aneurinolyticus, B. anthracia, B. aquaemaris, B.atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B.centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B.firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, Blarvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B.megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B.pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B.pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans,B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B.thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C.ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuriapenetrans, Pasteuria thornei, Pasteuria nishizawae, and Streptomycesspp. In a preferred embodiment, the spore is selected from the groupconsisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B.megaterium, and B. pumilus; and the germinative compound is selectedfrom the group consisting of L-alanine, L-valine, and L-asparagine.

In some embodiments of the aforementioned method, the germinativecompound is a polypeptide. In a preferred embodiment the germinativecompound in the aforementioned method is formulated, prior to drying, atconcentrations from 0.0001 mg/ml to 170 mg/ml. In some embodiments ofthe aforementioned method, the germinative compounds are formulated,prior to drying, at concentrations of 0.0003 mg/mL to 170 mg/mL, 0.0003mg/mL to 30 mg/mL, 0.001 mg/mL to 100 mg/mL, or 0.001 mg/mL to 10 mg/mL.In a preferred embodiment of the aforementioned method, the germinativecompound is formulated, prior to drying, at concentrations of from 0.001mg/mL to 1 mg/mL. In a further preferred embodiment of theaforementioned method, the spore has been shocked.

EXAMPLES

The following examples are intended to further illustrate the invention,but are not intended to limit the invention in any manner whatsoever.

In the following Examples, the terms “GOSD” and “GO+” refer tocompositions in which a germinative optimizer (L-alanine unlessspecified otherwise) was spray dried with the particular Bacillusspecies indicated. Spores of Bacillus species were spray dried withL-alanine being introduced to the spore mass immediately prior to spraydrying as a solution containing 0.044 grams of alanine per milliliter ofdistilled water.

The term “GO−” refers to compositions wherein the Bacillus species wassimilarly spray dried without a germinative compound being present.

Further, the following method was employed to determining sporegermination in the following Examples unless otherwise indicated. Whenspores are placed in nutrient solutions and begin to germinate theyrelease dipicolinic acid and ions which results in darkening. Thisindicator of germination results in a decrease in the optical extinctionof visible light by a spore suspension. The rate of germination wastherefore determined by counting the proportion of phase dark/brightspores, and monitoring the decrease in optical density at 600 nm (O.D.600) of germinating spore suspensions under a u.v.-visiblespectrophotometer. This is then converted to percent germination.

Example 1 Increased Germination of B. Subtilis ENV 923 in an IntimateMixture with L-Alanine

In order to compare the germination rate of the spores of intimatemixtures this invention with that of spores conventionally mixed with agerminant, the following treatments were performed:

A. Formation of an Intimate Mixture:

Spores of B. subtilis ENV 923 were spray dried with L-alanine beingintroduced to the spore mass immediately prior to spray drying as asolution containing 0.044 grams of alanine per milliliter of distilledwater. The intimate mixture produced was germinated by subsequentintroduction in a solution consisting of 0.01 M phosphate buffer indistilled water with resultant pH 7 and calibrated to a starting O.D.600 of 0.6.

B. Conventional Mixing of Spores with a Germinant:

Spores of B. subtilis ENV 923 were spray dried and subsequentlyintroduced into a solution consisting of 0.01 M phosphate buffer indistilled water with resultant pH 7. Spores were added to the buffersolution to calibrate to a starting O.D. 600 of 0.6. Alanine was addedto the solution at a concentration of 0.0001 grams of alanine permilliliter of solution.

C. Germination of Spores Alone:

Spores of B. subtilis ENV 923 were spray dried and subsequentlyintroduced into a solution consisting of 0.01 M phosphate buffer indistilled water with resultant pH 7 and calibrated to a starting O.D.600 of 0.6.

Two replications of each such treatment were performed. Table 1 belowshows the average results of such treatments affecting the germinationof B. subtilis ENV 923 as measured by a percent drop in optical density.A drop in optical density indicates progression of germination. Opticaldensity (OD) was measured at 600 nm wavelength with a Jenway Model 6320DVisible Range Spectrophotometer. L-alanine utilized was 99% purity andsourced from Alfa Aeser; Heysham, Lancashire, United Kingdom.

TABLE 1 Percent reduction from OD (600 nm) baseline over time. Sampletime (minutes) 0 5 10 15 20 Intimate 0% 2.7% 17.7% 34.3% 41.2% MixtureConventional 0% 0.5% 18.8% 31.9% 33.7% Mixing Spores 0% 1.3% 3.2% 4.1%7.5% Alone

The above results show that the spores in the intimate mixtures of thisinvention germinate more rapidly than do spores which are not intimatelymixed with the same germinant. Spore mixed with germinant exhibited muchgreater germination than spores alone.

Example 2 Increased Germination of Bacillus subtilis Strain ENV923Treated with GOSD

Bacillus subtilis spores were treated with GOSD via spray drying sporesin the presence of a solution of L-alanine and germination levels weredetermined via Optical Density (OD) readings.

0.01 M Potassium Phosphate Buffer, pH 7

The 0.01M Potassium Phosphate buffer was prepared using 1M K₂HPO₄ (87.09g dissolved in 0.5 L distilled water) and 1M KH₂PO₄ (68.045 g dissolvedin 0.5 L distilled water) solutions. Combining 61.5 ml of 1M K₂HPO₄ with38.5 ml 1 M KH₂PO₄ and diluting to 1000 ml with distilled water 0.1MPotassium Phosphate buffer at pH7.0 was made. Further diluting the 0.1MPotassium Phosphate buffer with distilled water at the ration 1:10, the0.01M Potassium Phosphate buffer, pH 7.0 was obtained. The buffer wassterilized by autoclaving at 121° C. for sixty (60) minutes.

Bacillus subtilis spore suspensions were prepared at the concentration1.7×10⁸ cfu/ml in 0.01 M Potassium Phosphate buffer, pH 7.0, incubatedin the preheated 37° C. water bath, and evaluated for percent ofgermination in 5 minute intervals over a 45 minute period.

TABLE 2 % Germination of Bacillus subtilis ENV923 at 37° C. with andwithout GOSD treatment Minutes 0 5 10 15 20 25 30 35 40 45 B. subtilisGOSD 0 13% 45% 63% 64% 75% 76% 80% 81% 83% B. subtilis Control 0  7%  9%10% 11% 14% 14% 16% 18% 19% Conclusion: GOSD treatment significantlyenhanced the percent germination and speed of germination of Bacillussubtilis spores.

Example 3 Increased Germination of Bacillus licheniformis Strain ENV100Treated with GOSD

Bacillus licheniformis spores were treated with GOSD via spray dryingspores in the presence of 0.044 grams of L-alanine per mL of distilledwater as described in Example 1. Germination levels were determined viaOptical Density (OD) readings as described above using 0.01 M PotassiumPhosphate buffer, pH 7.0 for spore suspension preparation.

Bacillus licheniformis spore suspensions were prepared at theconcentration 1.29×10⁸ cfu/ml in 0.01 M Potassium Phosphate buffer, pH7.0, incubated in the preheated 37° C. water bath, and evaluated forpercent of germination in five (5) minute intervals over a forty five(45) minute period.

TABLE 3 % Germination of Bacillus licheniformis treated and non-treatedspore suspensions Minutes 0 5 10 15 20 25 30 35 40 45 B. licheniformis,control 0 1% 5%  3% 10% 11% 10% 10% 10% 10% B. licheniformis, GOSD 0 4%5% 39% 60% 81% 85% 88% 91% 91% Conclusion: GOSD treatment significantlyenhanced percent germination and speed of germination of Bacilluslicheniformis spores.

Example 4 Increased Germination at Various pH Levels for Bacillussubtilis Strain ENV923 Treated with GOSD

Bacillus subtilis strain ENV923 GO+ and GO− spores were prepared asdescribed above and re-suspended in 0.01 M Potassium Phosphate buffer atvarious pH levels. OD₆₀₀ measurements were performed as described above.

0.01M Potassium Phosphate Buffers at pH 3.0-7.0

0.01M Potassium Phosphate buffer, pH 7.0 buffer was prepared asdescribed in Example 1.

-   -   To prepare 0.01M Potassium Phosphate buffers with pH range from        3.0 to 6.0 as base buffer was used 0.1 M Potassium Phosphate        buffer, pH 6.0 made by mixing 13.2 ml of 1M K₂HPO₄ and 86.8 ml        of 1M KH₂PO₄ solutions (described in Example 1) and bringing the        volume to 1 L with distilled water.    -   To obtain 0.01 M Potassium Phosphate buffers with pH 5.0 to pH        3.0, 0.1 M Potassium Phosphate buffer, pH 6.0 was diluted with        distilled water, pH of buffers was lowered to pH 5.0, pH 4.0 and        pH 3.0 using 1M H₂PO₄ and final volume brought up with distilled        water keeping ratio of 0.1 M buffer to distilled water 1:10.        Prepared buffers were stored at 4° C. and prior to each        experiment pH of buffers was re-adjusted using 1 M NaOH or 1 M        H₂PO₄.

TABLE 4 Germination results with GOSD (GO+)and without GOSD (GO−) atvarious pH levels, table shows percent germination measured at 5 minuteintervals. Minutes 0 5 10 15 20 25 30 35 40 45 GO+ pH 7 0 8% 48% 69% 80%84% 88% 90% 91% 92% GO− pH 7 0 3%  3%  8% 10% 11% 13% 14% 16% 16% GO+ pH6 0 4% 40% 58% 69% 75% 77% 78% 81% 82% GO− pH 6 0 1%  4%  5%  9%  8% 11%13% 12% 12% GO+ pH 5 0 7% 24% 34% 39% 41% 43% 45% 46% 46% GO− pH 5 0 4% 4%  5%  5%  6%  6%  7%  7%  7% GO+ pH 4 0 5% 23% 28% 30% 31% 33% 32%33% 34% GO− pH 4 0 3%  4%  5%  4%  4%  5%  3%  4%  4% GO+ pH 3.5 0 5%11% 16% 18% 18% 18% 18% 20% 20% GO− pH 3.5 0 2%  1%  1%  1%  1%  1%  2% 0%  1% GO+ pH 3 0 11%  16% 19% 19% 22% 21% 22% 21% 22% GO− pH 3 0 4% 4%  4%  5%  2%  3%  4%  2%  4% Conclusion: Treatment with GOSD enablesBacillus subtilis spores to germinate faster and overcome the effects oflower pH levels.

Example 5 Increased Germination of Bacillus subtilis Strain ENV923Treated with GOSD. Spore Germination Response as Affected by VariousTemperature Levels. Table Shows Percent Germination Measured at 10Minute Intervals

Bacillus subtilis strain ENV 923 GOSD and Control spores were preparedas described above. Germination of spores was tested via Optical Density(OD) measurements as described above. Spore suspensions for OD₆₀₀measurements were prepared and cooled to 4° C. in 0.01 M PotassiumPhosphate buffer, pH 7.0 (Phosphate buffer, pH 7.0 preparation) at theconcentration 1.7×10⁸ cfu/ml. For each spore suspension 3 culture tubesfilled to 3 ml volume were prepared. Following agitation and initialOD₆₀₀ measurement the tubes were incubated in water baths preheated to25° C., 30° C. and 37° C. for 120 min. At 10 min intervals tubes wereagitated and OD₆₀₀ measurements taken.

TABLE 5 Percent germination of Bacillus subtilis with GO− and GO+ at 37°C., 30° C. and 25° C. Minutes 0 10 20 30 40 50 60 70 80 90 100 110 120GO+, 37° C. 0 36%  58%  63% 68% 70% 70% 71% 70% 70% 70% 70% 70% GO+, 30°C. 0 12%  32%  45% 52% 55% 58% 59% 58% 60% 60% 60% 60% GO+, 25° C. 0 1%9% 16% 23% 28% 32% 33% 35% 37% 36% 35% 35% GO−, 37° C. 0 1% 4%  6%  7% 9%  9%  9%  9%  9%  9%  9%  9% GO−, 30° C. 0 0% 2%  3%  4%  6%  6%  7% 7%  8%  9%  8%  7% GO−, 25° C. 0 0% 2%  3%  4%  5%  7%  8%  8%  9% 10%10% 10% Conclusion: Treatment with GOSD (GO+) enables Bacillus subtilisspores to germinate faster and overcome the effects of lower temperatureregimes.

Example 6 Percent Germination of Bacillus licheniformis with and withoutGOSD in the Presence of Different Molar Solutions of NaCl Medium:

The medium was a dilute Tryptic Soy Broth (mTSB) (BD, 211822). Themedium was prepared by suspending 50 mg of Tryptic Soy Broth powder in 1L water with heat and agitation until completely dissolved. It was thenaliquoted into bottles and autoclaved for 30 minutes at 121° C. Based onthe manufacture's reported powder contents, the mTSB media contained perliter:

Pancreatic Digest of Casein: 28.3 mg Papic Digest of Soybean: 5.0 mgDextrose: 4.2 mg Sodium Chloride: 8.3 mg Dipotassium Phosphate: 4.2 mg

Sodium chloride (Amresco X190) was added to induce osmotic stress whereappropriate such that the final concentrations were 0.5 M or 1.5 M(29.22 g/L and 87.66 g/L respectively) before the media was heated andautoclaved.

Spore Suspensions:

Spore powders of Bacillus licheniformis strain ENV 100 treated or nottreated with GOSD were suspended in sterile water with 0.1% Octosol SLS(FT-SLS-246DRUM, Tiarco Chemical, Dalton, Ga.) within a sterile blenderjar. Spores were suspended by blending for 5 second intervals for atotal of at least 15 seconds or until the spores were completelysuspended visually. This was performed such that the final concentrationin the blender jar was 1×10¹⁰ cfu/ml. From this spore suspension, 250 μlwas transferred to tubes containing 4.75 ml mTSB to give a finalconcentration of 5×10⁸ cfu/ml. These concentrations are determined byoptimizations performed on each batch of spores to achieve a startingOD600 of approximately 0.6.

OD Germination Assay:

Tubes containing the suspended spores were immediately vortexed,measured at OD600 for time point zero, and incubated in a 37° C. waterbath. At respective time intervals, the time was recorded, tubes wereremoved, vortexed, measured at OD600 and returned to the water bath. Thepercent decrease in OD600 was determined by subtracting the measuredvalue from the zero time point, divided by the zero time point andmultiplied by 100%. Full germination was previously documented tocorrespond to a percent OD600 decrease of 60%. Therefore PercentGermination was determined by multiplying the percent OD600 decrease by1.67.

TABLE 6 % Germination of Bacillus licheniformis spores over one hourwith GOSD (GO+) and without (GO−) in the presence of 0, .5 Molar and 1.5Molar solutions of NaCl. GO+ GO− GO+ GO− GO+ GO− Time 0M 0M 0.5M 0.5M1.5M 1.5M 0:00 0 0 0 0 0 0 0:11  7% 0% 29% 1% 15% 2% 0:16 23% 0% 49% 0%30% 0% 0:22 44% 3% 61% 0% 44% 0% 0:27 53% 0% 67% 0% 48% 0% 0:32 59% 0%70% 1% 53% 0% 0:38 64% 0% 74% 1% 56% 0% 0:43 68% 0% 75% 0% 58% 0% 0:4971% 0% 77% 0% 61% 0% 0:55 72% 0% 77% 2% 63% 0% 1:01 75% 0% 78% 2% 66% 0%Conclusion: Treatment with GOSD enables Bacillus licheniformis spores togerminate faster and overcome the osmotic stress effects of various salt(NaCl) levels.

Example 7 Percent Germination of Bacillus licheniformis with and withoutGOSD in the Presence of Different Part Per Million Solutions of CopperMedium:

mTSB medium was prepared as above, but supplemented with NaCl to a finalconcentration of 50 mM to create an osmotically balanced media. A 1×10⁵ppm Copper (II) nitrate hemi(pentahydrate) (Alfa Aesar 12523) stocksolution was made by suspending 2 g into 20 ml water, and 0.22 μmfiltering. This was added to mTSB aliquots to achieve 0, 50, 100, and200 ppm final concentrations.

Spore Suspensions:

Spore powders of Bacillus licheniformis strain ENV 100 treated or nottreated with GOSD were suspended in sterile water with 0.1% Octosol SLS(FT-SLS-246DRUM, Tiarco Chemical, Dalton, Ga.) within a sterile blenderjar. Spores were suspended by blending for 5 second intervals for atotal of at least 15 seconds or until the spores were completelysuspended visually. This was performed such that the final concentrationin the blender jar was 2×10⁹ cfu/ml. From this spore suspension, 250 μlwas transferred to tubes containing 4.75 ml mTSB to give a finalconcentration of 1×10⁸ cfu/ml. These concentrations are determined byoptimizations performed on each batch of spores to achieve a startingOD600 of approximately 0.6.

OD Germination Assay:

Performed and calculated as indicated in Example 6.

TABLE 7 Percent germination of Bacillus licheniformis spores over onehour with GOSD (GO+) and without (GO−) in the presence of 0, 50 ppm, 100ppm and 200 ppm solutions of Copper ions. GO+ GO− GO+ GO− GO+ GO− GO+GO− Time 0 ppm 0 ppm 50 ppm 50 ppm 100 ppm 100 ppm 200 ppm 200 ppm 0:000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0:05 3.1% 0.6% 7.7% 1.5% 7.8%1.4% 5.0% 2.8% 0:11 19.8% 1.1% 14.3% 2.0% 12.5% 1.9% 9.0% 4.6% 0:1634.1% 2.2% 25.2% 2.0% 16.7% 3.3% 11.5% 4.6% 0:21 40.9% 4.4% 30.7% 1.5%19.3% 5.1% 14.0% 4.6% 0:26 47.7% 4.4% 34.0% 2.0% 18.2% 5.1% 15.0% 6.5%0:31 50.2% 4.4% 38.4% 1.5% 21.9% 5.6% 15.0% 5.6% 0:36 50.8% 4.4% 38.9%0.5% 20.8% 6.1% 17.0% 6.0% 0:41 52.0% 3.3% 40.6% 1.5% 21.4% 6.5% 18.0%5.6% 0:47 55.8% 3.9% 45.0% 3.0% 26.0% 7.5% 20.0% 6.0% 0:53 57.0% 2.8%44.4% 2.5% 25.5% 8.4% 19.5% 6.0% 0:58 57.6% 3.9% 45.5% 3.0% 25.0% 8.4%20.5% 6.9% 1:04 60.1% 4.4% 45.0% 1.0% 24.5% 8.4% 21.0% 4.6% Conclusion:Treatment with GOSD enables Bacillus licheniformis spores to germinatefaster and overcome the stress effects of various levels of Copper ions.

Example 8 Percent Germination of Bacillus licheniformis with and withoutGOSD in the Presence of Different Part Per Million Solutions of AluminumMedium:

mTSB medium was prepared as above, but supplemented with NaCl to a finalconcentration of 50 mM to create an osmotically balanced media. A 1000ppm Al³⁺ stock solution was made by suspending 0.62 g of Al₂(SO₄)₃*14H₂O (Alfa Aesar 12362) into 50 ml water, and 0.22 μm filtering. This wasadded to mTSB aliquots to achieve 0, 0.25, 0.50, and 1.0 ppm finalconcentrations. The pH of the media was then brought down to pH 4.5 bythe addition of HCl to allow complete separation of the Al³⁺ ion.

Spore Suspensions:

Spore powders of Bacillus licheniformis strain ENV 100 treated or nottreated with GOSD were suspended in sterile water with 0.1% Octosol SLS(FT-SLS-246DRUM, Tiarco Chemical, Dalton, Ga.) within a sterile blenderjar. Spores were suspended by blending for 5 second intervals for atotal of at least 15 seconds or until the spores were completelysuspended visually. This was performed such that the final concentrationin the blender jar was 1×10¹⁰ cfu/ml. From this spore suspension, 250 μlwas transferred to tubes containing 4.75 ml mTSB to give a finalconcentration of 5×10⁸ cfu/ml. These concentrations are determined byoptimizations performed on each batch of spores to achieve a startingOD600 of approximately 0.6.

OD Germination Assay:

Performed and calculated as in Example 6.

TABLE 8 Percent germination of Bacillus licheniformis spores over onehour with GOSD (GO+) and without (GO−) in the presence of 0, 0.25 ppm,0.50 ppm and 1.0 ppm solutions of Aluminum ions. GO+ GO− GO+ GO− GO+ GO−GO+ GO− Time 0 ppm 0 ppm 0.25 ppm 0.25 ppm 0.50 ppm 0.50 ppm 1.00 ppm1.00 ppm 0:00 0 0 0 0 0 0 0 0 0:07 21% 2%  9% 1% 26% 2% 30% 3% 0:14 48%3% 47% 4% 50% 4% 52% 3% 0:22 62% 3% 60% 5% 63% 4% 63% 4% 0:30 67% 2% 66%4% 67% 4% 67% 3% 0:37 70% 3% 68% 5% 70% 4% 70% 3% 0:44 71% 3% 69% 4% 71%4% 71% 3% 0:52 72% 3% 70% 4% 72% 5% 71% 3% 0:59 72% 3% 70% 5% 74% 6% 73%4% 1:07 72% 3% 72% 5% 74% 5% 72% 4% Conclusion: Treatment with GOSDenables Bacillus licheniformis spores to germinate faster and overcomethe stress effects of various levels of Aluminum ions.

Example 9 Percent Germination of Bacillus licheniformis with and withoutGOSD in the Presence of Different Millimolar Solutions of Bile SaltsMedium:

mTSB medium was prepared as above, but supplemented with NaCl to a finalconcentration of 50 mM to create an osmotically balanced media. A 80 mMbile salts stock solution was made by suspending 2.5 g sodiumtaurodeoxycholate (Sigma T0875), 1.1 g sodium glycodeoxycholate (SigmaG9910), and 0.346 g sodium deoxycholate (Sigma D5670) into 100 ml water,and 0.22 μm filtering. This was added to mTSB aliquots to achieve 0, 4,6, and 8 mM final concentrations.

Spore Suspensions:

Spore powders of Bacillus licheniformis strain ENV 100 treated or nottreated with GOSD were suspended in sterile water with 0.1% Octosol SLS(FT-SLS-246DRUM, Tiarco Chemical, Dalton, Ga.) within a sterile blenderjar. Spores were suspended by blending for 5 second intervals for atotal of at least 15 seconds or until the spores were completelysuspended visually. This was performed such that the final concentrationin the blender jar was 1×10¹⁰ cfu/ml. From this spore suspension, 250 μlwas transferred to tubes containing 4.75 ml mTSB to give a finalconcentration of 5×10⁸ cfu/ml. These concentrations are determined byoptimizations performed on each batch of spores to achieve a startingOD600 of approximately 0.6.

OD Germination Assay:

Performed and calculated as above.

TABLE 9 Percent germination of Bacillus licheniformis spores over onehour with GOSD (GO+) and without (GO−) in the presence of 0, 4 mM, 6 mMand 8 mM ppm solutions of Bile Salts. GO+ GO− GO+ GO− GO+ GO− GO+ GO−Time 0 mM 0 mM 4 mM 4 mM 6 mM 6 mM 8 mM 8 mM 0:00 0 0 0 0 0 0 0 0 0:0513% 4% 17% 1% 22% 1% 29%  7% 0:10 44% 5% 44% 1% 50% 2% 56% 10% 0:15 61%5% 61% 1% 62% 1% 67%  9% 0:20 70% 5% 69% 2% 70% 3% 73% 11% 0:26 76% 5%73% 2% 75% 2% 77% 11% 0:31 78% 5% 77% 3% 78% 3% 81% 11% 0:36 81% 5% 80%2% 80% 3% 83% 11% 0:41 83% 6% 82% 3% 82% 2% 84% 11% 0:46 85% 7% 83% 2%84% 2% 86% 11% 0:51 86% 6% 85% 2% 86% 3% 87% 11% 0:56 86% 6% 85% 2% 87%3% 88% 11% 1:01 87% 6% 87% 2% 88% 3% 90% 11% 1:06 88% 7% 89% 3% 89% 3%90% 11% Conclusion: Treatment with GOSD enables Bacillus licheniformisspores to germinate faster and overcome the stress effects of variouslevels of bile salts which can be encountered in a gastrointestinaltract.

Example 10 Average Growth of Three Replicates of Bacillus licheniformiswith or without GOSD in Defined Potassium Phosphate Medium and 2%Glucose

Bacillus licheniformis strain ENV 431 GO+ spores were treated with GOSD(procedure described earlier). As a control were used GO− spores fromthe same culture that were spray dried without using GOSD. The growthtest was performed in Minimal Salts Medium supplemented with 2% glucose.

Medium

The medium was prepared by dissolving (NH₄)₂SO₄ (1.26 g/L), MgCl₂ (0.81g/L), CaCl₂ (0.15 g/L), NaCl (0.05 g/L) in distilled water and adding 1ml/L 1000× Trace Mineral Mix (MnSO₄ (0.85 g/50 ml), ZnSO₄ (0.15 g/50ml), FeSO₄×7H₂O (0.15 g/50 ml), Thiamino-hydrochloride (0.05 g/50 ml).Prepared solution was poured into flasks (90 ml/flask) and autoclaved at121° C. for 40 min. Before inoculation the medium was supplemented with4 ml of filter sterilized solution of 25× Potassium Phosphate (K₂HPO₄(3.44 g/50 ml), KH₂PO₄ (2.81 g/50 ml)) and 2 ml of 50×(100 g/200 ml)glucose to 2% final concentration.

Growth of Bacillus licheniformis Cells

Amount of spores used for inoculation was determined using GO− and GO+spore powder counts. Concentrated (1000×) Bacillus licheniformis strainENV 431 spore suspensions were prepared by blending spores in thesterile blender jars using sterile water and added to the flasks withmedium to the concentrations indicated in the table below as 0 h. Thecounts of initial culture were obtained by performing dilutions andplate counts of blended spore suspensions. There were 3 flasks preparedfor each spore sample.

The flasks were incubated at 30° C., 150 rpm and grown for 48 h. Thesamples were taken and plate counts done at 24 h and 48 h.

TABLE 10 Average growth of three replicates of Bacillus licheniformiswith GOSD (GO+) or without GOSD (GO−)in minimal potassium phosphatemedium and 2% glucose. Data shown is in cfu/ml. 0 hour 24 hours 48 hoursB. licheniformis 1.88 × 10³ 2.20 × 10⁴  9.4 × 10⁴ GO− B. licheniformis1.46 × 10³ 7.95 × 10⁴ 3.65 × 10⁵ GO+ Conclusion: GOSD treatmentsignificantly enhanced Bacillus licheniformis germination and growthrate.

Example 11 Growth of Bacillus licheniformis Over a Two Day Period;Comparing Treatments with or without GOSD in the Presence of DifferentConcentrations of NaCl Medium:

mTSB was prepared as above, but with sodium chloride (Amresco X190)added to induce osmotic stress where appropriate such that the finalconcentrations were 0, 0.5, 1.0, or 1.5 M (0, 29.22, 58.44, or 87.66 g/Lrespectively). The media was heated, aliquoted to flasks, andautoclaved.

Plate Count Agar (PCA) (BD 247910) was prepared according to themanufacturer's instructions: 23.5 g of powder suspended in 1 L of water,bring to boil with frequent agitation, aliquot into glass jars, andautoclave. Media jars were cooled in a 45° C. water bath until needed.The manufacturer of the powder reports the following contents for PCAper liter:

Pancreatic Digest of Casein: 5.0 g Yeast Extract: 2.5 g Dextrose: 1.0 gAgar: 15.0 g Spore Suspensions:

Spore powders of Bacillus licheniformis strain ENV100 treated or nottreated with GOSD were suspended in sterile water with 0.1% Octosol SLS(FT-SLS-246DRUM, Tiarco Chemical, Dalton, Ga.) within a sterile blenderjar. Spores were suspended by blending for 5 second intervals for atotal of at least 15 seconds or until the spores were completelysuspended visually followed by serial dilutions in sterile water. Thiswas performed such that the final concentration in the culture flaskswas 1×10² cfu/ml.

Quantification:

Flasks were incubated at 37° C. shaking at 150 rpm for 28 hours (“1day”) or 50 hours (“2 days”). Aliquots from each flask were seriallydiluted into petri dishes with PCA cooled to <45° C. poured on top,swirled, and allowed to solidify. Plates were inverted and incubated forapproximately 24 hours at 37° C. Colonies were counted andconcentrations calculated based on dilutions. Approximately 10 μlsamples from each flask were also streaked on PCA plates to test forpurity.

TABLE 11 Growth of Bacillus licheniformis treated with GOSD (GO+) whenchallenged by osmotic stress from salt solution. Day GO+ or− Molarcfu/ml 1 GO+ 0 1.05E+06 1 GO− 0 7.33E+03 1 GO+ 0.5 3.60E+06 1 GO− 0.55.00E+06 1 GO+ 1.0 3.00E+05 1 GO− 1.0 2.31E+05 1 GO+ 1.5 1.22E+04 1 GO−1.5 1.50E+02 2 GO+ 0 8.17E+06 2 GO− 0 4.90E+05 2 GO+ 0.5 7.03E+06 2 GO−0.5 8.43E+06 2 GO+ 1.0 2.07E+06 2 GO− 1.0 3.50E+06 2 GO+ 1.5 4.33E+05 2GO− 1.5 1.53E+02 Conclusion: GOSD treatment significantly enhancedgermination and growth of Bacillus licheniformis under osmotic saltstress.

Example 12 Protease Activity of Bacillus subtilis Strain ENV 923

Bacillus subtilis strain ENV 923 spores treated with GOSD (GO+) asdescribed earlier, and non-treated (GO−) were used for the proteaseactivity test.

Medium

Chemically Defined Salt Medium (CDSM) was used for cell propagation in aprotease test. Medium was prepared by dissolving base solutioncomponents (g/L): (NH₄)SO₄, 1.26 g; L-glutamic acid, 1.18 g; MgCl₂,0.81; CaCl₂, 0.155 and 85% L-lactic acid (0.530 ml/L) in distilled waterand adding 1 ml/L of 1000× Trace Mineral Mix (g/50 ml). Flasks with basesolution (48 ml/flask) were autoclaved for 40 min. Before inoculation 2ml of separately prepared filter sterilized 25× buffer solution withglucose (g/50 ml): MOPS, 11.6; KH₂PO₄, 0.6; glucose, 4.5 were added.

Growth of Bacillus subtilis ENV923 Cells

The amount of spores used for inoculation was determined using GO− andGO+ spore powder counts. Concentrated (1000×) Bacillus subtilis strainENV923 spore suspensions were prepared by blending spores in the sterileblender jars using sterile 0.01 M Potassium Phosphate buffer, pH 7.0 andadded to the flasks at the concentration of about 1×10⁴ cfu/ml. Thecounts of initial culture were confirmed by performing dilutions andplate counts of blended spore suspensions. There were 3 flasks preparedfor each spore sample.

The flasks were incubated at 30° C., 150 rpm, and samples were taken at48 h.

Protease Activity Assay

Protease activity assay was carried out on cell supernatants usingcasein as substrate and Folin & Ciocalateu's Phenol reagent that reactswith tyrosine and facilitates blue color development. Protease activityunit was defined as amount of enzyme that liberates 1 μg of tyrosine inone minute. Amount of tyrosine in test tubes was determined by measuringOD₆₅₀ in a Jenway 7305 spectrophotometer and calculating liberatedtyrosine using a standard curve.

Reagents Reagent 1: 0.05 M Potassium Phosphate Buffer, pH 7.0

0.1 M Potassium Phosphate buffer, pH 7.0 (prepared as described inExample 1) was diluted to at the ratio 1:1 with distilled water toobtain 0.05 M Potassium Phosphate buffer, pH 7.0

Reagent 2: 0.65% Casein Solution

0.65 g of casein were dissolved in 80 ml of 0.05 M Potassium Phosphatebuffer, pH 7.0, heated to bring casein into solution, and the finalvolume brought to 100 ml with 0.05 M Potassium Phosphate buffer, pH 7.0.

Reagent 3: 15% Trichloroacetic Acid (TCA)

15 g of TCA were dissolved in distilled water and the final volumebrought to 100 ml.

Reagent 4: 20% Na₂CO₃

20 g of Na₂CO₃ dissolved in distilled water and final volume brought to100 ml.

Protease Assay

-   -   10 ml of culture were centrifuged and supernatant filtered        through 0.2 μm filter into sterile tubes.    -   3 ml of filtered supernatant were mixed with 3 ml of 0.65%        casein solution and put into 37° C. water bath for 1 h.    -   Reaction was stopped by adding 6 ml of 15% TCA and samples        centrifuged for 5 min.    -   0.5 ml of each sample was mixed with 1 ml of 20% Na₂CO₃,        followed by 0.5 ml of Folin & Ciolcallieu's Phenol reagent        addition and incubation for 20 min at room temperature to allow        blue color development.    -   3 ml of distilled water were added to each sample and after        mixing the OD₆₅₀ was measured.    -   To calculate protease activity the standard curve for tyrosine        was prepared obtaining dilution series of tyrosine dissolved in        distilled water, treating them to the same conditions as the        culture samples, and measuring OD₆₅₀.

TABLE 12 Protease production of Bacillus subtilis treated with GOSD(GO+) versus control (GO−) Bacillus subtilis Protease activity units 54h. GO+ 71.5 GO− 40 Conclusion: Treatment of Bacillus subtilis sporeswith GOSD enables greater production of enzymes such as protease.

Example 13 Germination of Streptomyces viridochromogenes in the Presenceof Germinative Compounds

Streptomyces viridochromogenes spores were harvested from plates bypouring 10 ml of TX buffer (0.05 M Tris-HCl buffer, pH 7.3 with 0.001%Tween 80) and removing the spores with a sterile cotton swab. Sporesuspensions from plates were poured into sterile 50 ml tubes. When sporesuspensions of all samples were obtained, heat shock was performed byputting the tubes with spore suspensions into a heat block, allowing thetemperature to reach 55° C., and maintain the temperature at 55° C. for10 min. After heat shock spore suspensions were cooled in ice water for5 min and spun down for 30 min. Supernatant was poured off, sporesre-suspended in 25 ml 0.02 M Potassium Phosphate buffer, pH 7.0 at 4° C.and spun down for 15 min. After pouring off supernatant, spores werere-suspended in 20 ml of 0.02 M Potassium Phosphate buffer, pH 7.0 andvigorously mixed to obtain the spore suspension that was used in theexperiment.

Samples were prepared by mixing 1.5 ml of 2× germinant blend with 1.5 mlspore suspension. All germinant blends were prepared in distilled wateras 2×50 ml solutions. Calcium chloride was prepared as a 100× solution(0.4 g/10 ml) and 20 μl were added to 10 ml of 2× germinant blends. Thefinal concentrations of the germinative compounds were as follows: 0.89mg/ml of L-alanine; 1.17 mg/ml of L-valine, 13.2 mg/ml of L-asparagine;2.25 mg/ml of glucose; and 2.25 mg/ml of fructose. After measuringinitial OD600, samples were transferred to a 30° C. water bath and OD600was measured at 15 min intervals for 90 min to determine germinationrates.

TABLE 13 ENV 151 (Streptomyces viridochromogenes) % Reduction in OpticalDensity Ion/Germinant treatment 0 15 30 45 60 75 90 0.01M KPO4 0 1.0%2.0% 5.4% 6.2% 4.9% 5.4% 0.01M KPO4, CaCl₂ 0 7.3% 3.4% 6.1% 6.1% 8.0%9.3% 0.01M KPO4, CaCl₂, L-Ala 0 1.8% 5.2% 12.2% 11.9% 14.2% 17.6% 0.01MKPO4, CaCl₂, L-Val 0 2.0% 4.9% 11.2% 7.8% 14.4% 18.0% 0.01M KPO4, CaCl₂,L-Asn 0 2.0% 6.1% 8.8% 11.7% 8.0% 9.0% 0.01M KPO4, CaCl₂, L-Ala, L-Asn 07.7% 12.1% 14.4% 19.8% 20.4% 18.6% 0.01M KPO4, CaCl₂, L-Ala, L-Asn,glucose 0 8.2% 11.7% 14.5% 20.1% 19.4% 20.7% 0.01M KPO4, CaCl₂, glucose0 7.0% 8.8% 7.0% 10.5% 12.6% 13.2% 0.01M KPO4, CaCl₂, glucose, fructose0 5.9% 8.3% 12.0% 12.7% 14.6% 14.6% 0.01M KPO4, CaCl₂, L-Ala, L-Asn,glucose, fructose 0 11.7% 14.0% 17.7% 20.4% 22.2% 22.4% Conclusion:Treatment of Streptomyces viridochromogenes spores with germinativecompounds enhanced germination.

Example 14 Comparison of Germination Rates Between Intimate Mixtures andConventional Mixing of Bacterial Spores and Germinative Compounds

In order to compare the germination rate of the spores of intimatemixtures with that of spores conventionally mixed with a germinant, thefollowing treatments are performed:

A. Formation of an Intimate Mixture:

Spores of B. subtilis, B. amyloliquefaciens, B. brevis, B. cereus, B.coagulans, B. firmus, B. laterosporus, B. licheniformis, B. megaterium,B. mycoides, B. popilliae, B. polymyxa, B. pumilus, B. thuringiensis,Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae,Streptomyces viridochromogenes, Streptomyces griseoviridis, Streptomyceslydicus, Streptomyces plicatus, Streptomyces sindeneusis, Streptomycesrochei, Streptomyces alni, Streptomyces viridis, Streptomycesthermovulgaris, Streptomyces griseus, Streptomyces acidiscabies,Steptomyces aureofaciens, Streptomyces galbus, Streptomyces microflavus,and Streptomyces aureofacien are dried with L-alanine, L-valine,L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagineor L-phenylalanine being introduced to the spore mass immediately priorto drying as a solution containing 0.044 grams of the amino acid permilliliter of distilled water. The intimate mixture produced isgerminated by subsequent introduction in a solution consisting of 0.01 Mphosphate buffer in distilled water with resultant pH 7.

B. Conventional Mixing of Spores with a Germinant:

Spores of B. subtilis, B. amyloliquefaciens, B. brevis, B. cereus, B.coagulans, B. firmus, B. laterosporus, B. licheniformis, B. megaterium,B. mycoides, B. popilliae, B. polymyxa, B. pumilus, B. thuringiensis,Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae,Streptomyces viridochromogenes, Streptomyces griseoviridis, Streptomyceslydicus, Streptomyces plicatus, Streptomyces sindeneusis, Streptomycesrochei, Streptomyces alni, Streptomyces viridis, Streptomycesthermovulgaris, Streptomyces griseus, Streptomyces acidiscabies,Steptomyces aureofaciens, Streptomyces galbus, Streptomyces microflavus,and Streptomyces aureofacien are hydrated and dried. Such spores aregerminated by the introduction in a solution consisting of 0.01 Mphosphate buffer in distilled water with resultant pH 7, and 0.0001grams of L-alanine, L-valine, L-proline, L-leucine, L-cysteine,L-threonine, L-glutamine, L-asparagine or L-phenylalanine per milliliterof solution.

C. Germination of Spores Alone:

Spores of B. subtilis, B. amyloliquefaciens, B. brevis, B. cereus, B.coagulans, B. firmus, B. laterosporus, B. licheniformis, B. megaterium,B. mycoides, B. popilliae, B. polymyxa, B. pumilus, B. thuringiensis,Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae,Streptomyces viridochromogenes, Streptomyces griseoviridis, Streptomyceslydicus, Streptomyces plicatus, Streptomyces sindeneusis, Streptomycesrochei, Streptomyces alni, Streptomyces viridis, Streptomycesthermovulgaris, Streptomyces griseus, Streptomyces acidiscabies,Steptomyces aureofaciens, Streptomyces galbus, Streptomyces microflavus,and Streptomyces aureofacien are hydrated and dried. The spores aresubsequently introduced into a solution consisting of 0.01 M phosphatebuffer in distilled water with resultant pH 7.

The germination of spores resulting for each such treatment is measuredby the percent drop in optical density or by counting the number ofgerminated spores under a microscope. It is found that the intimatelymixed compositions germinate more rapidly than their correspondingconventionally mixed equivalent.

What is claimed is:
 1. A dried intimate mixture comprising a bacteriaspore and a germinative compound, wherein the bacteria spore and thegerminative compound are maintained in proximate position until theyreach an environment conducive to germination.
 2. The mixture of claim 1wherein the spore is selected from the group consisting of B.alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B.anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B.caldolyyicus, B. centrosporus, B. cereus, B. circulans, B. clausii, B.coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B.infernus, B larvae, B. laterosporus, B. lentus, B. lentimorbus, B.licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B.mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B.pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus,B. sporothermodurans, B. stearothermophilus, B. subtilis, B.thermoglucosidasius, B. thuringiensis, B. vulgatis, B.weihenstephanensis, C. thermocellum, C. ljungdahlii, C. acetobutylicum,C. beijerinckii, and C. butyricum.
 3. The mixture of claim 1 wherein thegerminative compound is selected from the group consisting of L-alanine,L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine,L-asparagine, L-phenylalanine and analogues thereof.
 4. The mixture ofclaim 1 wherein said composition is a mixture selected from the groupconsisting of L-alanine+Bacillus subtilis, L-alanine+Bacilluslicheniformis, L-alanine+Bacillus pumilus, L-alanine+Bacillusamyloliquefaciens, L-alanine+Bacillus coagulans, L-alanine+Bacilluscereus, L-alanine+Bacillus clausii, L-alanine+Clostridium butyricumL-valine+Bacillus subtilis, L-valine+Bacillus licheniformis,L-valine+Bacillus pumilus, L-valine+Bacillus amyloliquefaciens,L-valine+Bacillus coagulans, L-valine+Bacillus cereus, L-valine+Bacillusclausii, L-valine+Clostridium butyricumL-alanine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillusamyloliquefaciens, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus coagulans, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus cereus, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus clausii, L-alanine+glucose+fructose+potassium ions(GFK)+Clostridium butyricum L-asparagine+glucose+fructose+potassium ions(GFK)+Bacillus subtilis, L-asparagine+glucose+fructose+potassium ions(GFK)+Bacillus licheniformis, L-asparagine+glucose+fructose+potassiumions (GFK)+Bacillus pumilus, L-asparagine+glucose+fructose+potassiumions (GFK)+Bacillus amyloliquefaciens,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus cereus,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus clausii,L-asparagine+glucose+fructose+potassium ions (GFK)+Clostridium butyricumL-alanine+inosine+Bacillus subtilis, L-alanine+inosine+Bacilluslicheniformis, L-alanine+inosine+Bacillus pumilus,L-alanine+inosine+Bacillus amyloliquefaciens, L-alanine+inosine+Bacilluscoagulans, L-alanine+inosine+Bacillus cereus, L-alanine+inosine+Bacillusclausii, L-alanine+inosine+Clostridium butyricumL-proline+glucose+Bacillus megaterium, L-proline+Bacillus megaterium,and L-lactate+Clostridium butyricum.
 5. The mixture of claim 1 whereinsuch mixture is produced by spray-drying or freeze-drying.
 6. Themixture of claim 1 wherein the spore is selected from the groupconsisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B.megaterium, and B. pumilus; and the germinative compound is selectedfrom the group consisting of L-alanine, L-valine, and L-asparagine.
 7. Acomposition comprising the dried intimate mixture of claim
 1. 8. Thecomposition of claim 7 wherein the spore is selected from the groupconsisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B.aneurinolyticus, B. anthracia, B. aquaemaris, B. atrophaeus, B.boronophilus, B. brevis, B. caldolyyicus, B. centrosporus, B. cereus, B.circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B.fusiformis, B. globigii, B. infernus, B larvae, B. laterosporus, B.lentus, B. lentimorbus, B. licheniformis, B. megaterium, B.mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus,B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B.schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B.stearothermophilus, B. subtilis, B. thermoglucosidasius, B.thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C.ljungdahlii, C. acetobutylicum, C. beijerinckii, and C. butyricum. 9.The composition of claim 7 wherein the germinative compound is selectedfrom the group consisting of L-alanine, L-valine, L-proline, L-leucine,L-cysteine, L-threonine, L-glutamine, L-asparagine, L-phenylalanine andanalogues thereof.
 10. The composition of claim 7 wherein saidcomposition is a mixture selected from the group consisting ofL-alanine+Bacillus subtilis, L-alanine+Bacillus licheniformis,L-alanine+Bacillus pumilus, L-alanine+Bacillus amyloliquefaciens,L-alanine+Bacillus coagulans, L-alanine+Bacillus cereus,L-alanine+Bacillus clausii, L-alanine+Clostridium butyricum,L-valine+Bacillus subtilis, L-valine+Bacillus licheniformis,L-valine+Bacillus pumilus, L-valine+Bacillus amyloliquefaciens,L-valine+Bacillus coagulans, L-valine+Bacillus cereus, L-valine+Bacillusclausii, L-valine+Clostridium butyricumL-alanine+glucose+fructose+potassium ions (GFK)+Bacillus subtilis,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus licheniformis,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillus pumilus,L-alanine+glucose+fructose+potassium ions (GFK)+Bacillusamyloliquefaciens, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus coagulans, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus cereus, L-alanine+glucose+fructose+potassium ions(GFK)+Bacillus clausii, L-alanine+glucose+fructose+potassium ions(GFK)+Clostridium butyricum L-asparagine+glucose+fructose+potassium ions(GFK)+Bacillus subtilis, L-asparagine+glucose+fructose+potassium ions(GFK)+Bacillus licheniformis, L-asparagine+glucose+fructose+potassiumions (GFK)+Bacillus pumilus, L-asparagine+glucose+fructose+potassiumions (GFK)+Bacillus amyloliquefaciens,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus coagulans,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus cereus,L-asparagine+glucose+fructose+potassium ions (GFK)+Bacillus clausii,L-asparagine+glucose+fructose+potassium ions (GFK)+Clostridium butyricumL-alanine+inosine+Bacillus subtilis, L-alanine+inosine+Bacilluslicheniformis, L-alanine+inosine+Bacillus pumilus,L-alanine+inosine+Bacillus amyloliquefaciens, L-alanine+inosine+Bacilluscoagulans, L-alanine+inosine+Bacillus cereus, L-alanine+inosine+Bacillusclausii, L-alanine+inosine+Clostridium butyricumL-proline+glucose+Bacillus megaterium, L-proline+Bacillus megaterium,and L-lactate+Clostridium butyricum.
 11. The composition of claim 7wherein such mixture is produced by spray-drying or freeze-drying. 12.The composition of claim 7 wherein such composition further comprises anenteric coating.
 13. The composition of claim 7 wherein the spore isselected from the group consisting of B. subtilis, B. amyloliquefaciens,B. licheniformis, B. megaterium, and B. pumilus; and the germinativecompound is selected from the group consisting of L-alanine, L-valine,and L-asparagine.
 14. A method for preparing a dried intimate mixturecomprising a bacteria spore and a germinative compound, the methodcomprising: a) preparing a solution comprising a bacteria spore and agerminative compound; and b) drying the solution to obtain a driedintimate mixture comprising a bacteria spore and a germinative compound,wherein the bacteria spore and the germinative compound are maintainedin proximate position until they reach an environment conducive togermination.
 15. The method of claim 14, wherein the drying isspray-drying or freeze-drying.
 16. The method of claim 14, wherein thespore is selected from the group consisting of B. alcalophilus, B.alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracia, B.aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus,B. centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B.firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, Blarvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B.megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B.pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B.pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans,B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B.thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C.ljungdahlii, C. acetobutylicum, C. beijerinckii, and C. butyricum. 17.The method of claim 14, wherein the spore is selected from the groupconsisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B.megaterium, and B. pumilus; and the germinative compound is selectedfrom the group consisting of L-alanine, L-valine, and L-asparagine. 18.A dried intimate mixture produced by the method of claim
 14. 19. Amethod for increasing the germination of bacteria spores, comprising: a)preparing a solution comprising a bacteria spore and a germinativecompound; and b) drying the solution to obtain a dried intimate mixturecomprising a bacteria spore and a germinative compound, wherein thebacteria spore and the germinative compound are maintained in proximateposition until they reach an environment conducive to germination of thebacterial spores; and c) exposing the intimate mixture to an environmentconducive to germination of the bacteria spores, wherein the germinationof the bacteria spores in the intimate mixture is increased relative toa corresponding bacteria spore formulation that lacks a germinativecompound.